Assessment of a PK/PD Target of Continuous Infusion Beta-Lactams Useful for Preventing Microbiological Failure and/or Resistance Development in Critically Ill Patients Affected by Documented Gram-Negative Infections

Background: Emerging data suggest that more aggressive beta-lactam PK/PD targets could minimize the occurrence of microbiological failure and/or resistance development. This study aims to assess whether a PK/PD target threshold of continuous infusion (CI) beta-lactams may be useful in preventing microbiological failure and/or resistance development in critically ill patients affected by documented Gram-negative infections. Methods: Patients admitted to intensive care units from December 2020 to July 2021 receiving continuous infusion beta-lactams for documented Gram-negative infections and having at least one therapeutic drug monitoring in the first 72 h of treatment were included. A receiver operating characteristic (ROC) curve analysis was performed using the ratio between steady-state concentration and minimum inhibitory concentration (Css/MIC) ratio as the test variable and occurrence of microbiological failure as the state variable. Area under the curve (AUC) and 95% confidence interval (CI) were calculated. Independent risk factors for the occurrence of microbiological failure were investigated using logistic regression. Results: Overall, 116 patients were included. Microbiological failure occurred in 26 cases (22.4%). A Css/MIC ratio ≤ 5 was identified as PK/PD target cut-off with sensitivity of 80.8% (CI 60.6–93.4%) and specificity of 90.5% (CI 74.2–94.4%), and with an AUC of 0.868 (95%CI 0.793–0.924; p < 0.001). At multivariate regression, independent predictors of microbiological failure were Css/MIC ratio ≤ 5 (odds ratio [OR] 34.54; 95%CI 7.45–160.11; p < 0.001) and Pseudomonas aeruginosa infection (OR 4.79; 95%CI 1.11–20.79; p = 0.036). Conclusions: Early targeting of CI beta-lactams at Css/MIC ratio > 5 during the treatment of documented Gram-negative infections may be helpful in preventing microbiological failure and/or resistance development in critically ill patients.


Introduction
The global increase of antimicrobial resistance represents a major health concern [1]. Although beta-lactams are still the backbone of treatment for the management of Gramnegative infections in critically ill patients [2], it should be mentioned that the incidence of resistance is rapidly increasing [3].
Beta-lactams exhibit short elimination half-life and time-dependent pharmacodynamics (PD); their efficacy is related to the percentage of the dosing interval in which the free plasma concentration is maintained above the minimum inhibitory concentration (MIC) of the bacterial pathogen (%fT >MIC ) [4]. Consensus is lacking regarding methods for identifying a pharmacokinetic/pharmacodynamic (PK/PD) target that may maximize the effectiveness of beta-lactams in the treatment of Gram-negative infections among critically ill patients. According to experimental animal models, the minimum threshold needed to achieve bactericidal activity with beta-lactams is 40-70% fT >MIC [5], namely, a target that has been adopted in pivotal trials of novel antimicrobial agents [6,7]. However, emerging clinical data suggest that more aggressive PK/PD targets up to 100%fT >4-5×MIC may give rise to better outcomes in critically ill patients [6,8,9].
Optimizing beta-lactam pharmacodynamics could be a means by which to overcome resistance development [10][11][12]. Preclinical studies with different beta-lactams (namely, piperacillin-tazobactam, meropenem, and ceftazidime) showed that trough concentration (C min )/MIC ratios ranging between 3.8 and 6.2 may be helpful in preventing the emergence of resistance among Gram-negatives [13][14][15]. Continuous infusion (CI) may represent the best administration mode for maximizing the pharmacodynamics of beta-lactams under the same daily dose.
The aim of this study was to assess whether a PK/PD target threshold of continuous infusion (CI) beta-lactams may be useful in preventing microbiological failure and/or resistance development in critically ill patients affected by documented Gram-negative infections.

Patient Population, Microbiological Characteristics, and TREATMENT Regimens
From December 2020 to July 2021, a total of 526 patients who underwent therapeutic drug monitoring (TDM)-guided beta-lactam therapy in our hospital were screened. Among them, 116 were selected and included in the study (52, 45, and 19 receiving meropenem, piperacillin/tazobactam, and ceftazidime or ceftazidime/avibactam, respectively; see Figure 1). Demographics and clinical characteristics of the included patients are reported in Table 1.
The global increase of antimicrobial resistance represents a major health concern [1]. Although beta-lactams are still the backbone of treatment for the management of Gramnegative infections in critically ill patients [2], it should be mentioned that the incidence of resistance is rapidly increasing [3].
Beta-lactams exhibit short elimination half-life and time-dependent pharmacodynamics (PD); their efficacy is related to the percentage of the dosing interval in which the free plasma concentration is maintained above the minimum inhibitory concentration (MIC) of the bacterial pathogen (%fT>MIC) [4]. Consensus is lacking regarding methods for identifying a pharmacokinetic/pharmacodynamic (PK/PD) target that may maximize the effectiveness of beta-lactams in the treatment of Gram-negative infections among critically ill patients. According to experimental animal models, the minimum threshold needed to achieve bactericidal activity with beta-lactams is 40-70% fT>MIC [5], namely, a target that has been adopted in pivotal trials of novel antimicrobial agents [6,7]. However, emerging clinical data suggest that more aggressive PK/PD targets up to 100%fT> 4-5× MIC may give rise to better outcomes in critically ill patients [6,8,9].
Optimizing beta-lactam pharmacodynamics could be a means by which to overcome resistance development [10][11][12]. Preclinical studies with different beta-lactams (namely, piperacillin-tazobactam, meropenem, and ceftazidime) showed that trough concentration (Cmin)/MIC ratios ranging between 3.8 and 6.2 may be helpful in preventing the emergence of resistance among Gram-negatives [13][14][15]. Continuous infusion (CI) may represent the best administration mode for maximizing the pharmacodynamics of beta-lactams under the same daily dose.
The aim of this study was to assess whether a PK/PD target threshold of continuous infusion (CI) beta-lactams may be useful in preventing microbiological failure and/or resistance development in critically ill patients affected by documented Gram-negative infections.
Significantly higher microbiological failure and/or resistance development was observed in patients with beta-lactam C ss /MIC ≤ 5 compared to those with C ss /MIC > 5 (21/30 vs. 5/86; p < 0.001; Figure 3). Table 2 summarizes the results of a multivariate regression analysis that assessed possible factors associated with microbiological failure and/or resistance development. C ss /MIC ratio ≤ 5 of CI beta-lactams (odds ratio [OR] 34.54; 95%CI 7.45-160.11; p < 0.001) and Pseudomonas aeruginosa infections (OR 4.79; 95%CI 1.11-20.79; p = 0.036) were shown, through multivariate regression analyses, to be independent predictors of microbiological failure and/or resistance development of Gram-negatives among critically ill patients.  Relationship between beta-lactam PK/PD target attainment (Css/MIC) and microbiological failure and/or resistance development. Each point represents a single critically ill patient treated with selected beta-lactams. Dotted line represents optimal PK/PD cut-off (Css/MIC = 5) identified at ROC analysis. Significantly higher risk of microbiological failure and/or resistance occurrence was found in patients with a Css/MIC below the optimal cut-off (p < 0.001). Css: steadystate concentrations.   Relationship between beta-lactam PK/PD target attainment (Css/MIC) and microbiological failure and/or resistance development. Each point represents a single critically ill patient treated with selected beta-lactams. Dotted line represents optimal PK/PD cut-off (Css/MIC = 5) identified at ROC analysis. Significantly higher risk of microbiological failure and/or resistance occurrence was found in patients with a Css/MIC below the optimal cut-off (p < 0.001). Css: steadystate concentrations.  . Relationship between beta-lactam PK/PD target attainment (C ss /MIC) and microbiological failure and/or resistance development. Each point represents a single critically ill patient treated with selected beta-lactams. Dotted line represents optimal PK/PD cut-off (C ss /MIC = 5) identified at ROC analysis. Significantly higher risk of microbiological failure and/or resistance occurrence was found in patients with a C ss /MIC below the optimal cut-off (p < 0.001). C ss : steady-state concentrations.

Discussion
To the best of our knowledge, this is the first real-world study that identified significant association between C ss /MIC threshold of CI beta-lactams and microbiological failure and/or resistance development among critically ill patients affected by documented severe Gram-negative bacterial infections.
The finding of a C ss /MIC ratio ≤ 5 as a strong independent predictor of microbiological failure stresses the relevance that this threshold may have, not only for maximizing clinical efficacy, but also for minimizing the development of resistance [6,10]. This is in agreement with the recommendations of the international guidelines for the management of critically septic patients [9,16]. In our study, it is noteworthy that only a minority (approximatively 5%) of the patients who achieved a PK/PD target above this threshold (namely a 100%T > 5 × MIC ) within the first 72h experienced microbiological failure or underwent breakthrough resistance.
Our findings are consistent with those of some preclinical models [13][14][15], showing that PK/PD targets required for suppressing the emergence of beta-lactam resistance should be higher compared to those required for clinical efficacy. A dynamic in vitro hollow fiber infection model showed that when using meropenem, ceftazidime, or cefepime in intermittent infusion, a C min /MIC ratio > 3.8 may be helpful in suppressing the development of resistance of Pseudomonas aeruginosa or Klebsiella pneumoniae [13]. In another dynamic in vitro hollow fiber infection model in which intermittent infusion piperacillin-tazobactam was used, it was shown that a C min /MIC ratio of 4.6 allowed for resistance suppression when dealing with a relatively low bacterial inoculum of P. aeruginosa (4 × 10 5 CFU/mL) [15]. However, when dealing with a much larger P. aeruginosa bacterial load (8 × 10 8 CFU/mL), the same threshold neither caused any significant bacterial killing nor suppressed the emergence of resistance, [15]. In another dynamic in vitro hollow fiber infection model with intermittent infusion meropenem, it was shown that a C min /MIC ratio > 6.2 allowed to suppress resistance development of Pseudomonas aeruginosa and that the needed threshold was 4-fold lower when meropenem was combined with aminoglycosides [14]. Consistent with these findings, some authors considered that combination therapy of meropenem plus an aminoglycoside could be helpful for treating Gram-negative infections and for suppressing the emergence of resistance in the presence of a high bacterial burden, as is commonly the case in VAP [11,17]. However, the role of combination therapy is not supported by our analysis, that showed no benefit of combo therapy compared to beta-lactam monotherapy in preventing microbiological failure or resistance development. Conversely, our analysis was in agreement with the findings of a recent retrospective observational multicenter study of ceftazidime/avibactam, which showed no benefit of combination therapy compared to monotherapy [18].
Notably, the achievement of a specific PK/PD threshold with CI compared to intermittent infusion may show remarkable advantages for beta-lactams. The time-dependent PD activity coupled with the short elimination half-lives make this administration mode suitable. Advantages may include administration of lower doses, minimization of fluctuations in antibiotic serum levels, and avoidance of high peak concentrations commonly reported with intermittent infusion and potentially associated with the occurrence of toxicity (e.g., neurotoxicity) [7,19,20].
Infections caused by Pseudomonas aeruginosa also emerged as an independent risk factor for microbiological failure and/or for resistance development in this study. It should not be overlooked that in our study, approximately half of the critically ill patients who had microbiological failure and/or resistance development were affected by Pseudomonas aeruginosa infection. Pseudomonas aeruginosa infection has been identified as a significant predictor of high resistance rate, especially when treatment is based on piperacillin/tazobactam or meropenem [21,22]. The use of extended-infusion (EI) and/or CI may represent an effective strategy for preventing the development of resistance with beta-lactams. Interestingly, EI of ceftolozane/tazobactam, by allowing the achievement of higher PK/PD target compared to intermittent infusion, has emerged as a protective factor in terms of resistance development in patients affected by carbapenem-resistant Pseudomonas aeruginosa [23]. Consequently, we believe that in the treatment of critically ill patients affected by Pseudomonas aeruginosa infections, the use of high-doses CI beta-lactams, focused on achieving an early, aggressive PK/PD target of C ss /MIC > 5, may represent a valuable approach for suppressing resistance development and preventing the emergence of MDR/XDR Pseudomonas aeruginosa colonization. This approach may be even more relevant when dealing with challenging pathophysiological conditions (e.g., augmented renal clearance) and/or with deep-seated infections (e.g., pneumonia) [7,24,25]. In this regard, it should not be overlooked that the bacterial burden in pneumonia is usually higher compared to that observed in other sources of infection (e.g., urinary tract infections). This inoculum effect in VAP may attenuate the effectiveness of beta-lactams [19]. Notably, piperacillin-tazobactam and the antipseudomonal cephalosporins were shown to be especially prone to the inoculum effect in Pseudomonas aeruginosa infections [26]. Consequently, the identification of more aggressive PK/PD targets is mandatory in VAP, as suboptimal exposure may potentially favor the development of resistance.
Our analysis was limited only to the old beta-lactams, but we are confident that the same principles could be applied to the novel agents (e.g., ceftolozane-tazobactam, ceftazidime-avibactam, meropenem-vaborbactam, cefiderocol) as well. Novel beta-lactams represent the last resort for the management of carbapenem-resistant Enterobacterales and/or MDR/XDR Pseudomonas aeruginosa or Acinetobacter baumannii, and the definition of which PK/PD target threshold should be granted for preventing microbiological failure and for avoiding the development of resistance is one of the major clinical issue that must be addressed in the next few years [11].
We are aware of some limitations in our study. The limited sample size and the retrospective, monocentric study design should be acknowledged. The analysis was based on total beta-lactam concentrations. However, considering the low plasma protein binding (ranging from <10% for meropenem and ceftazidime and approximately 20-30% for piperacillin), no relevant impact on C ss /MIC ratio calculation would be expected. The analysis did not take into account the role of beta-lactamase inhibitors (i.e., tazobactam and avibactam). However, it should be mentioned that no definite PK/PD indexes for the beta-lactamase inhibitors were established in preclinical models for resistance suppression. The presence of combination therapy and of polymicrobial infections could be potential confounders, but they occurred only in a minority of cases (<25%). Finally, MIC values were determined by automated testing methods and not through broth microdilution.
In conclusion, this is the first real-world study to have identified a significant association between C ss /MIC threshold of CI beta-lactams and microbiological failure and/or resistance development in Gram-negative infections. Both microbiological failure and/or resistance development in Gram-negative infections could be prevented by the early achievement of an aggressive PK/PD target of Css/MIC > 5 during treatment with CI beta-lactams in critically ill patients. Further prospective studies are warranted in order to confirm these findings and identify whether this PK/PD index could be applied to novel beta-lactams as well.

Patients
All the critically ill patients admitted to the general intensive care unit (ICU), transplant ICU, or COVID ICU of the IRCCS Azienda Ospedaliero-Universitaria in Bologna from December 2020 to July 2021 who were treated with beta-lactams because of suspected or documented Gram-negative infections were retrospectively retrieved. Inclusion criteria were: (1) use of piperacillin-tazobactam, ceftazidime, ceftazidime-avibactam, or meropenem by continuous infusion (CI) for at least 72 h; (2) TDM performed in the first 72 h after starting treatment; (3) isolation of Gram-negative pathogens from microbiological cultures and determination of susceptibility for the specific beta-lactam (namely punctual MIC value).

Beta-Lactam Administration and Sampling
Selected beta-lactams were prescribed at the discretion of the treating physician or infectious disease consultant in terms of therapeutic indication, dosage, and duration according to current clinical practice implemented at the IRCCS Azienda Ospedaliero-Universitaria in Bologna. For all the selected beta-lactams, a loading dose (LD), (2 g for meropenem and ceftazidime, 2.5 g for ceftazidime-avibactam and 9 g for piperacillintazobactam) was administered over 2-h infusion. Maintenance dose (MD) was administered by CI (q6-8 h infused over 6-and 8-h for meropenem and ceftazidime-avibactam due to stability restrictions; over 24-h for piperacillin-tazobactam and ceftazidime according to stability in aqueous infusion [27]), and dosing regimens were selected at the discretion of the treating physician or infectious disease consultant according to renal function and underlying pathophysiological conditions. Antibiotics 2021, 10, 1311 9 of 12 Blood samples were collected in the first 72 h from the beginning of antibiotic treatment in order to determine beta-lactam C ss . Total blood concentrations of piperacillin, ceftazidime, and meropenem were measured at the hospital Unique Metropolitan Laboratory concentrations were analyzed by means of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) commercially available method (Chromsystems Instruments & Chemicals GmbH, Munich, Germany) and were provided available for clinical review within 6 h from blood collection.
Combination therapy was defined as the concomitant use with a beta-lactam of other antibiotics active against Gram-negatives (namely aminoglycosides, colistin, fosfomycin, fluoroquinolones, and tigecycline).

Data Collection
Demographic (age, sex, weight, height, body mass index [BMI]) and clinical/laboratory data (need for mechanical ventilation and vasopressors, implementation of continuous renal replacement therapy [CRRT] at baseline, creatinine clearance, presence of augmented renal clearance [ARC], site/type of infection, isolated pathogens, MIC, genetic mechanism of resistance, beta-lactam dosing, C ss at the first TDM assessment, implementation of antibiotic combination therapy, treatment duration, occurrence and timing of relapse, resistance development) were collected for each included patient.

Microbiological and Susceptibility Data
C ss /MIC ratio was calculated for each patient at that first TDM assessment that was always performed within 72 h from starting treatment. Gram-negative pathogens were isolated from various infection sites: blood, bronchoalveolar lavage, peritoneal fluid, urine, cerebrospinal fluid, and tissue biopsies. For BAL and urine culture a gram-negative bacterial load ≥10 4 and ≥10 5 CFU/mL was considered significant, respectively [28]. Genetic analysis was performed in case of isolation of carbapenem-resistant Enterobacterales. Carbapenemase type was determined by multiplex immunochromatographic assay NG test CARBA 5 (NG Biotech, Guipry-Messac, France) for detecting the specific carbapenemase enzyme produced (IMP, VIM, NDM, KPC, OXA-48). In patients having multiple Gramnegative isolates, C ss /MIC ratio was calculated using the higher MIC value. The MIC of the identified Gram-negative pathogens was determined by means of E-test methodology, and interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints.
Microbiological failure was defined as the persistence of the same gram-negative pathogen isolated from index culture after ≥7 days from starting beta-lactam treatment, as previously reported [29]. Resistance development was defined as the increase of the MIC of the clinical isolate beyond the EUCAST clinical breakpoint. Microbiological eradication was defined as the presence of negative cultures in at least two subsequent assessments.

Statistical Analysis
Descriptive statistics were used to describe the patient sample, with continuous data presented as median and IQR, while categorial variables were expressed by count or percentage.
The ROC curve analysis was performed using the C ss /MIC ratio as the test variable and emergence of relapse/resistance as the state variable, and AUC along with 95%CI was calculated. The optimal cut-off point was computed using the Youden Index method. Youden Index was calculated according to the following equation: sensitivity (%) + specificity (%) − 100.
Univariate comparisons between patients who experienced microbiological failure and those who did not were performed by the Fisher's exact test or the Chi-Square test. All the independent covariates with a p value of <0.05 at the univariate analysis were included in a multivariate logistic regression model. A p value of <0.05 was considered significant.